Novel Members of the AKH/RPCH Peptide Family...

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1 Novel Members of the /RPCH Peptide Family Pestycydy, 2007, (3-4), ISSN Novel Members of the /RPCH Peptide Family: Isolation of from the Corpora Cardiaca of the Two Beetle Species, Cheilomenes Lunata and Coccinella Septempunctata Susanne NEUPERT Friedrich-Schiller University, Institut of Zoology, Erbertstrasse 1, Jena, Germany Abstract: Adipokinetic hormones () are synthesized in endocrine cells of the glandular corpora cardiaca, in the anterior part of the retrocerebral complex. plays an important role in the metabolism of carbohydrates that fuel energydepleting activities such as flight and walking. To date, the primary sequences of eight isoforms from various beetle species have been elucidated. In this study, the glandular part of the CC from two Ladybird beetles, Cheilomenes lunata and Coccinella septempunctata were directly analysed by MALDI-TOF mass spectrometry. Subsequently, the amino acid sequences of both putative novel s were confirmed by tandem mass spectrometry. Keywords: adipokinetic hormons (s), MALDI-TOF mass spectrometry, insect, beetles INTRODUCTION Members of the adipokinetic hormon/red pigment-concentrating hormon family are exclusively produced in glandular cells of the retrocerebral complex which occupy, in most insects, the anterior part of the corpora cardiaca (CC). They are not synthesized in neurons of the nervous system, and thus, they are not neuropeptides [1]. The first was isolated from the CC glands of locusts where it displays lipid-mobilising activity and fuel production for long-distance flight [2]. All s are characterized by (1) N-terminally blocked pyroglutamate,

2 40 S. Neupert (2) eight to ten amino acids, (3) at least two aromatic amino acids, as well as (4) overall mainly uncharged molecule [3, 4]. s exert a wide range of diverse effects [4] such a regulation of carbohydrates and lipid metabolism, as well as, effectors for energy-costly activities [5, 6]. In beetles, like in other insects, proline is the amino acid that powers energy consuming activities as flight, walking and ball-rolling [6]. More than 30 different members of the family were identified from various invertebrates [4]. In this study, the primary sequence of two putative novel s from the European Ladybird beetle Coccinella septempunctata and the Southern African Ladybird beetle Cheilomenes lunata were identified by means of direct MALDI- TOF mass spectrometry. De novo sequencing yielded amino acid sequences similar to known of Tribolium castaneum [7]. MATERIAL AND METHODS Animals. Ladybird beetles, Cheilomenes lunata were caught in ( ) Namibia by Dr. Reinhard Predel and Coccinella septempunctata were caught in Jena (Germany) by the author. Sample preparation for mass spectrometry. The glandular area of the corpora cardiaca (CC) was dissected and prepared for MALDI-TOF mass spectrometric analysis as described by Predel [8]. MALDI-TOF mass spectrometry. Mass spectra in the mass range of Da were acquired in positive ion mode on a Bruker Daltonic ultraflexii TOF/TOF biospectrometry workstation (Bruker Daltonic GmbH, Bremen, Germany) equipped with a pulsed nitrogen laser. Laser strength was adjusted to provide an optimal signal-to-noise ratio. For the tandem MS experiments, the protonated PDF mass ion signal was selected and fragmented by tandem mass spectrometry (CID: collision-induced dissociation). The fragmentation data obtained in these experiments was handled using the Protein Prospector ( RESULTS AND DISCUSSION Two novel octapeptide members of the /RPCH family have been identified from the glandular part of the corpora cardiaca (CC) of two species of Ladybird beetles by means of direct MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry (Figure 1.). The majority

3 Novel Members of the /RPCH Peptide Family of have no charge which is represented by a characteristic ion signature [M+Na] + and [M+K] + in resulting mass spectra [8, 9]. Thus, the putative of both beetle species are observed as [M+Na] + : Da and [M+K] + : Da. By using tandem mass spectrometry techniques, the same samples were subjected to CID (collision-induced dissociation). The manual analysis of the fragment series resulted in the sequence pqlnftpnwamide, with a monoisotopic mass at [M+H] + : Da (Table 1; Figure 2). Comparing with s from other beetle species, the both ladybird beetle s, designated as Chl- (Cheilomenes lunata) and Cos- (Coccinella septempunctata), shows sequence conformity with Trc- of Tribolium cataneum (Table 1). An additional similarity of these three s is an amino acid modification at position 5 (Thr) which could be of interest for phylogenetic studies. Signal intensity 1.3E [M+Na] [M+Na] [M+K] MS MS Cheilomenes lunata CC Coccinella septempunctata CC [M+K] m/z Figure 1. MALDI-TOF mass spectra of CC-preparations of Cheilomenes lunata and Coccinella septempunctata in a mass range of Da. The arrow marks the ion signals of the putative in both beetle species.

4 42 S. Neupert b pq L N F T P N W y W N P T F N LQp 1.3E+4 Signal Intensity F N L W y y2 a2 b y3 a3 b y4 a y5 a5 b5 y a6 b pqlnftpnwa [M+Na] + : m/z 1100 Figure 2. Representative CID spectrum of the ion signal at of Cheilomenes lunata which represents a Na + alkali cation ion signal ([M+Na] + ) of the putative Chl-. A number of y- and b-type fragment ions are labelled. The fragments were analysed manually and the resulting sequence is given in the inset. The CID spectrum of Cos- is identical and hence not shown. Table 1. Comparison of primary structure of peptides of the /RPCH family in various beetle species. Similarities are bold labelled Species -1-2 References peptide sequence [M+H] + peptide [M+H] + m/z sequence m/z Decapotoma lunata pqlnfspnwgna pqlnfspnwa [10] Onitis aygulus pqynfstgwa pqfnyspdwa [11] Gareta nitens pqfnyspvwa pqfnyspdwa [12] Scarabaeus deludens pqfnyspvwa pqfnyspdwa [12] Geotrupes stercorosus pqlnyspdwa [13] Pachnoda marginata pqlnyspdwa [14] Melolontha melolontha pqlnyspdwa [13] Tenebrio molitor pqlnfspnwa [15] Zophobas rugipes pqlnfspnwa [15] Tribolium castaneum pqlnftpnwa [7] Leptinotarsa decemlineata pqltftpnwa pqvnfspnwa [14] Coccinella septempunctata pqlnftpnwa this study Cheilomenes luncata pqlnftpnwa this study

5 Novel Members of the /RPCH Peptide Family Acknowledgments I acknowledge the financial assistance of the Deutsche Forschungsgemeinschaft (PR 595/6-1, 2). I will also thank R. Predel (Jena) for critical discussions of the manuscript, and O. Scheibner (HKI, Department of Biostructure Chemistry Jena, Germany) for supporting tandem MALDI-TOF measurements. REFERENCES [1] Predel R., Cockroach neuropeptides: Sequences, localisation, and physiological actions, in: Invertebrate Neuropeptides and Hormones: Basic Knowledge and Recent Advances, 2006, pp [2] Stone J.V., Mordue W., Batley K.E. Morris H.R., Nature, 1976, 263, [3] Gäde G., Z. Naturforsch, 1996, 51c, [4] Gäde G., Hoffmann K.H., Spring J.H., Physiol. Rev., 1997, 77, [5] Goldsworthy G.J., The endocrine control of flight metabolism in locusts. Academic Press, New York 1983, pp [6] Gäde G., Auerswald L., Comp. Biochem. Physiol. B, 2002, 132, [7] Li B., Predel R., Neupert S., Hauser F., Tanaka Y., Verleyen P., Cazzamali G., Williamson M., Schoofs L., Schachtner J., Grimmelikhuijzen C., Park Y., 2007, Genome Res., in press. [8] Predel R., J. Comp. Neurol., 2001, 436, [9] Konig S., Albers C., Gäde G., Rapid Commun. Mass Spectrom., 2005, 19, [10] Auerswald L., Gäde G., J. Exp. Biol., 1995, 198, [11] Gäde G., Biochem. J., 1997, 321, [12] Gäde G., Biochem. Biophys. Res. Commun., 1997, 230, [13] Gäde G., Biochem. J., 1991, 275, [14] Gäde G., J. Comp. Physiol., 1989, 159, [15] Gäde G., Rosiński G., Peptides, 1990, 11,

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